It remains evident that most injuries caused by surgery, trauma or disease heal by fibrosis and formation of scar tissue. Therapy to stimulate regeneration of musculoskeletal tissue in situ would alleviate much of the emotional and economic burden associated with imperfect therapies, but mammalian epimorphic regeneration continues to be an elusive target. Multiple labs, including our own, have shown that immune cells are essential for regeneration where they orchestrate the injury response. Identifying specific immune cell subpopulations that regulate regeneration and how they direct resident cells to polarize the healing response are key areas of research. The long-term goal of our research is to understand the mechanistic basis for mammalian musculoskeletal regeneration in order to catalyze therapies that inhibit fibrosis and stimulate regeneration where it does not naturally occur. The objectives of this proposal are to characterize regulatory T (TREG) cell phenotypes associated with regenerating and scarring microenvironments, functionally test the role of TREG cells for regeneration, and to test how T cell paracrine factors drive local fibroblast behavior. Our central hypothesis is that a regeneration-specific population of TREG cells are required for musculoskeletal regeneration. TREG cells represent fundamental dominant-negative immune cells that coordinate inflammation. Despite the importance of TREG cells during the inflammatory response, how they contribute to regeneration in mammals remains unknown. Completing Aim 1 will enhance our understanding of whether TREG cells have regeneration-specific phenotypes through single-cell RNAseq. Additionally, it will test the functional requirement of these cells for musculoskeletal regeneration by generating a transgenic spiny mouse. Completing Aim 2 will illuminate how secreted T cell factors regulate fibroblast behavior and drive a regenerative phenotype by performing intra- and inter-species co-cultures of isolated T cells and primary fibroblasts. The rationale for pursuing this work is that these results will provide the necessary foundation for an in-depth interrogation to test how specific T cell interactions and the factors they produce regulate blastema formation in mammals.